Background and Objective:Enterococcus faecalis offers challenges when present in an anatomically challenging and inaccessible site such as root canal and are instrumental in treatment failures. This emphasizes the need for irrigants besides mechanical instrumentation in root canal treatment to reinstate tooth structure and function. We explored the efficacy of chlorine dioxide and lemon extracts as alternatives to the conventional irrigant sodium hypochlorite on E. faecalis. Materials and Methods: The anti-enterococcal activity of the test and control irrigants was detected by disk diffusion method. The time taken by the chlorine dioxide, lemon extract (undiluted and 1:1 dilution), sodium hypochlorite, and saline to produce enterococcicidal activity was estimated by contact time studies, and the colony counts of E. faecalis at the time intervals of 1, 5, 10, 30, and 60 min and 24 h following exposure with irrigants were determined. Results: Chlorine dioxide and undiluted lemon extract demonstrated better anti-enterococcal activity than conventional irrigant sodium hypochlorite. Complete enterococcicidal activity was noted after a time interval of 1 min of exposure with chlorine dioxide, undiluted lemon extract, and sodium hypochlorite and after 10 min of exposure with 1:1 diluted lemon extract. Conclusion: The nonentity in literature on studies assessing the contact time of chlorine dioxide solution and lemon extract for their possibility as root canal irrigant gave an impetus for the study. Chlorine dioxide and undiluted lemon extract exhibited bactericidal activity against E. faecalis within 1 min of exposure equivalent to the activity of customary irrigant sodium hypochlorite, thus validating their possibility to be used as root canal irrigants.

Primary teeth often demand for endodontic treatment than permanent teeth due to high incidence of carious lesion. Pulpal therapy of deciduous dentition is mandatory to retain the structural and functional role of teeth besides providing psychological benefits to the patients.[1]Enterococcus faecalis, a Gram-positive cocci noted for its resilient properties, offers challenges when present in an anatomically challenging and inaccessible site such as root canal and are instrumental in treatment failures.[2] Effective elimination of microbes is the key to success of any endodontic treatment. This emphasizes the need for irrigants with antimicrobial, more specifically anti-enterococcal activity besides mechanical irrigation.[3]

Sodium hypochlorite is a customary root canal irrigant employed at concentrations of 0.5%–5.25% and is also noted for appreciable toxicity and carcinogenic properties.[4] Chlorine dioxide exists as gas in water. Its excellent microbicidal activity at wide pH ranging from 3 to 9, along with little or no side effects, has conferred it as a choice among root canal irrigants during recent times.[5],[6] Among other factors for the inclusion of a chemical for its efficacy as a root canal irrigant, the contact time required to produce bactericidal activity should be factored.[7]

As chemical agents induce side effects, research is attempted to seek herbal alternatives for their easy availability, cost-effective, low toxicity, and bactericidal properties. Citrus has the above-said benefits.[8] We recognized the nonentity in literature on studies assessing the contact time of chlorine dioxide solution and lemon extract for their possibility as root canal irrigant.

The present study was aimed to evaluate the anti-enterococcal activity and contact time required for chlorine dioxide solution and lemon extract to inhibit E. faecalis and compare them with the activity of conventional root canal irrigant sodium hypochlorite.

Materials and Methods

Determining the inhibitory activity of irrigants against Enterococcus faecalis

Preparation of test irrigants

The concentration and the compounds used as test and control irrigants in the study are presented in [Table 1].

Table 1: Concentration and grouping of the irrigants employed in the study

Chlorine dioxide solution was prepared by dissolving one chlorine dioxide tablet (Lifesystems) in 10 ml of distilled water to make a concentration of 300 ppm as instructed by the manufacturer. The solution was freshly prepared at the time of use.

Lemon extract

Fresh lemon fruits were obtained from the local market at Chidambaram. The fruits were washed in running tap water, rinsed with sterile distilled water, and cut open with a sterile knife and the extract obtained by squeezing was filtered (using Whatman filter paper No. 1) to remove the seeds and pulp. The fresh extract was diluted by mixing 5 ml of fresh lemon extract with 5 ml of sterile distilled water to obtain 1:1 diluted lemon extract.

Sodium hypochlorite

The chemical was obtained commercially (Chloraxid) and used as per established protocol.

Sterile saline

Saline solution was prepared by addition of 900 mg of sodium chloride (Fisher Scientific, Qualigens, India) to 100 ml of distilled water and sterilized by autoclaving.

Bacterial inoculum

A single colony of E. faecalis (ATCC 29212) from an overnight culture on blood agar was aseptically inoculated on sterile brain–heart infusion broth and incubated at 37°C for 4 h in aerobic environment. Following incubation, the suspension was quality checked by Gram staining. This suspension was adjusted to match the turbidity of a McFarland 0.5 standard (1.5 × 108 colony-forming unit [CFU] ml − 1) and used as inoculum.

Disk diffusion procedure

The inhibitory activity of the irrigants against E. faecalis was determined using Mueller–Hinton agar plates (Hi-Media, India). The bacterial inoculum was spread using a sterile applicator to obtain a lawn culture on the surface of dry Mueller–Hinton agar plate. Sterile disks of 6 mm diameter were placed on inoculated petri plates and 10 μl of each irrigant was applied to each disk drop wise using a micropipette. Upon drying, the plate was incubated aerobically for 24 h and the zone of inhibition obtained around the irrigants was measured in mm. Experiment was performed in triplicates and the mean value for the inhibitory zone obtained was analyzed and compared with one-way ANOVA test and Student's t-test using SPSS (Statistical Package for the Social Sciences) software 16 version. P < 0.05 at 5% level of significance was considered statistically significant.

Determining the contact time required by irrigants to inhibit Enterococcus faecalis

The time required by irrigants to produce an inhibitory activity on E. faecalis was assessed at 6 time intervals viz., 1, 5, 10, 30, and 60 min and 24 h.[7] Test was performed using 24-well cell culture plates (Tarson) each labeled appropriately for the irrigants and time intervals of the study. To the labeled wells of the plate, 1 ml of E. faecalis inoculum adjusted to 0.5 McFarland standard was added and mixed with 1 ml of test irrigant at concentration mentioned in [Table 1]. After the elapse of the study time period, 1 ml of the suspension from each well was aseptically transferred into sterile tubes containing 2 ml of the neutralizer. The purpose of using the neutralizer was to prevent the continued action of the irrigants against E. faecalis. The neutralizer used for sodium hypochlorite was 0.6% sodium thiosulfate and sterile saline served as neutralizer for the other irrigants of the study. Aliquots from these tubes were studied for qualitative analysis of irrigant activity and reduction in colony counts of E. faecalis following exposure to irrigants at varying time intervals.

Qualitative analysis of irrigants activity on Enterococcus faecalis

Following the study time exposure of bacterial suspension to irrigants, the presence of any viable E. faecalis was determined by spot inoculating the aliquot from the neutralizer-containing tubes onto the surface of nutrient agar plates. The plate was incubated aerobically for 37°C for 24 h and checked for the presence or absence of colonies. The purity of the colonies obtained was further assessed by standard microbiologial procedures including Grams stain, catalase test, bile esculin hydrolysis, salt and heat tolerance test.[9]

The activity of irrigants as either bactericidal or bacteriostatic, on E. faecalis, was determined by the colony counts obtained on spread plate method. About 500 μl of the suspension from tubes containing the neutralizer along with bacterial suspension exposed to irrigants were serially diluted. Aliquots from the tubes were transferred to the surface of dry nutrient agar plates and spread evenly using a sterile L rod. The plates were incubated at 37°C aerobically for 24 h and the colonies formed were counted and calculated to obtain the CFU/ml.

Results

Determining the inhibitory activity of irrigants against Enterococcus faecalis by disk diffusion method

The disk diffusion assay demonstrated the zone of inhibition measuring 14 mm and 11 mm for chlorine dioxide and undiluted lemon extract, respectively. The activity of 1:1 diluted lemon extract was lesser than sodium hypochlorite. [Table 2] shows the statistical details for the inhibitory activity of irrigants using one way ANOVA analysis.

Statistically significant observations were demonstrated by chlorine dioxide when compared with other irrigants indicating their high antibacterial efficacy. However, the activity of undiluted lemon extract when compared with sodium hypochlorite was not statistically significant [Table 3].

Determining the contact time required by irrigants to inhibit Enterococcus faecalis

Qualitative analysis of irrigants activity on Enterococcus faecalis

The irrigants, chlorine dioxide, undiluted lemon extract, and sodium hypochlorite, demonstrated complete inhibition of E. faecalis following contact time of 1, 5, 10, 30, and 60 min and 24 h. Growth of E. faecalis was noted with diluted lemon extract after a contact time of 1 and 5 min. Sterile saline at all the contact time periods studied demonstrated growth of E. faecalis[Figure 1].

Mechanical instrumentation of primary teeth is regarded complicated due to bizarre internal geometry, furcational connections, and horizontal anastomoses, thus leaving certain areas untouched. Therefore, to aid in complete debridement of necrotic tissue and disinfection of infected root canal, the need of an irrigant becomes imperative.[10],[11]E. faecalis is a normal flora of the oral cavity, which, by virtue of its survival advantage and virulence factors proficiently, invades and survives in dentinal tubules, and is associated with 63% of necrotic primary teeth.[12]

Sodium hypochlorite is the most commonly used root canal irrigant encompassing twin benefits of antimicrobial and tissue dissolving properties. These physiognomies have conferred sodium hypochlorite as a “gold standard” for irrigation.[13] The concentration of 5.25% of sodium hypochlorite as intracanal irrigant associated with hand and rotary instrumentation was found effective.[14] However, its corrosive action on metals, unpleasant taste, toxicity to periradicular tissues, and ability to induce permanent damages to tooth follicles, peripheral tissues, and oral mucosa deflates their utility.[11] Hence, a search for alternative irrigants was sought.

Chlorine dioxide with its strong antimicrobial properties, ease of use, and widespread availability is gaining popularity as a root canal irrigant. It is similar to chlorine and hypochlorite, but differs by being a true gas in solutions, nontoxic up to 3000 ppm, nonirritant and noncarcinogen.[5] The paucity in literature on the contact time studies for its use as irrigant propelled us in this dimension of the study.

As an attempt to elude from the repercussion of antibiotic usage for pathogens which exhibit multiple drug resistance, an extensive search is made toward herbal therapy. Lemon was preferred as test irrigant because of its easy availability and for the simplicity in its extract preparation. Freshly prepared extract of plant products is always better than commercially available products, as no preservatives are added in the former one which may give biased results.[15] Lemon besides an antimicrobial agent plays a paramount role in collagen synthesis, immune activation, osteogenesis, and wound healing.[16] In our study, the inhibitory zone produced employing a volume of 10 μl of test irrigants viz., chlorine dioxide (13. 66 mm), undiluted lemon extract (11.33 mm), and diluted lemon extract (10.33 mm) against E. faecalis was found to be greater than inhibitory zone produced by the conventional irrigant sodium hypochlorite (8. 33 mm). Chlorine dioxide exerts antimicrobial activity by virtue of its powerful oxidizing property which disrupts the transport of substances across the cell wall of bacteria.[17] The antibacterial activity of lemon juice might be due to the acidic pH that could affect the charges of the amino acids that constitute the peptidoglycan and active site of enzymes.[18] Sodium hypochlorite exerts antibacterial activity by targeting the bacterial enzymatic sites causing irreversible inactivation, biosynthetic alterations in cellular metabolism, and phospholipid destruction.[19]

Bactericidal activity of chemical agent could be presented by finding the time taken (contact time) to produce negative cultures, which signify the 100% growth inhibition of microorganisms in the presence of irrigants.[20] The effectiveness of an irrigating solution depends on the nature of the organism and the time of its contact with organism.[21] As no irrigant can be kept in continuous contact with dentin for longer duration, the present study checked the efficiency of the irrigant at 1, 5, 10, 30, and 60 min and 24 h time intervals.

In our study, complete inhibition of E. faecalis growth following a contact time of 1 min with test irrigants chlorine dioxide and undiluted lemon extract was observed similar to the conventional irrigant sodium hypochlorite. A study by Gomes et al., employing 5.25% of sodium hypochlorite, inhibited bacterial growth in a contact time of 1 min. A study performed using high-purity chlorine dioxide on planktonic E. faecalis showed chlorine dioxide killed all E. faecalis and delivered the best result after 1 min in the absence of dentin powder.[22] The antibacterial effect of chlorine dioxide and sodium hypochlorite was found to be higher than chlorhexidine in eliminating intracanal biofilms of E. faecalis on extracted human teeth.[5]

In our study, lemon extract was found to have inhibitory activity against E. faecalis analogous to other commercially available irrigants. The inhibitory activities of medicinal herbs, hydroalcoholic extract of aloe vera, and hydroalcoholic extract of garlic against E. faecalis had been reported earlier.[15],[23]

Conclusion

The anti-enterococcal activity of chlorine dioxide and undiluted lemon extract was found comparable to the activity of conventional root canal irrigant sodium hypochlorite. In addition, contact time required by these test irrigants to produce bactericidal activity was found to be <1 min. Hence, on the basis of enterococcicidal activity, this study endorses chlorine dioxide and undiluted lemon extract as contemporary root canal irrigants.

Acknowledgments

We acknowledge the technical assistance rendered by Mr. R. Venkatesan, Department of Microbiology, Rajah Muthiah Dental College and Hospital.